Development of the testis cell transplantation technique established a system to study the biology of spermatogonial stem cells (SSCs) as well as the differentiation processes that occur during spermatogenesis. Currently there are no biochemical, molecular or morphological criteria by which the SSC can be identified, and only a functional assay can establish the presence of the SSC in any cell population. During the past eight years the transplantation technique has demonstrated that the stem cell can be cryopreserved, transplanted from many species, maintained in vitro, and subjected to genetic modification. The technique also has been used to establish the cell type (germ cell or Sertoli cell) responsible for both natural and induced genetic mutations, and to demonstrate that cell cycle timing during spermatogenesis is controlled by the genetic program of the germ cell. Thus, the transplantation system has provided a powerful approach to study the stem cell, its niche in the seminiferous tubule, and the process of spermatogenesis. Despite these advances, it is not possible to obtain and study pure populations of SSCs from testes of mature animals, which greatly impedes studies on the biology of these cells. We propose a series of experiments in the mouse to further enrich testis cell populations for stem cell content and then to use these purified populations for introduction of genetic modifications into the SSCs. An important group of genes we plan to examine for their effect are those that have been shown to immortalize stem cells. This approach will provide us with two populations of germ cells: 1) testis cell populations isolated directly from the animal and greatly enriched for stem cells, and 2) immortalized cell lines that can be propagated in vitro as stem cells or early stage spermatogonia. The ability of these cells to colonize the seminiferous tubules of recipient animals and generate spermatogenesis as well as their ability to differentiate in vitro will be used to characterize the original cell populations and to study the differentiation process. The transplantation technique has been a productive approach in our studies over the past eight years and has proven increasingly valuable to other scientists. The studies outlined will continue progress toward our objective of understanding the biology of SSCs and spermatogenesis, and many of our findings will be applicable to other species.